Tert promoter mutations in cancer

ABSTRACT

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions related to certain promoter mutations in cancer. In one embodiment, a method for treating a subject having thyroid cancer comprises the steps of (a) obtaining a biological sample from the subject; (b) performing an assay on the sample obtained from the subject to identify a mutation at 1 295 228 C&gt;T (C228T) and 1 295 250 C&gt;T (C250T), corresponding to −124 C&gt;T and −146 C&gt;T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene; (c) identifying the subject as having or likely to develop aggressive thyroid cancer if the C228T and/or C250T mutation is identified; and (d) treating the subject with one or more treatment modalities appropriate for a subject having or likely to develop aggressive thyroid cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/780,600, filed Sep. 28, 2015, which is a 35 U.S.C. § 371 U.S.national entry of International Application PCT/US2014/031967, having aninternational filing date of Mar. 27, 2014, which claims the benefit ofU.S. Provisional Application No. 61/833,773, filed Jun. 11, 2013, andU.S. Provisional Application 61/805,710, filed Mar. 27, 2013.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.RO1CA134225, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of cancer. More specifically,the present invention provides methods and compositions related tocertain promoter mutations in cancer.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P12422-03_ST25.txt.” The sequence listing is 1,605 bytes in size, andwas created on Mar. 27, 2014. It is hereby incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Thyroid cancer is the most common classical endocrine malignancy, andits incidence has been rising rapidly in the US as well as otherindustrialized countries over the past few decades. Thyroid cancers areclassified histologically into four groups: papillary, follicular,medullary, and undifferentiated or anaplastic thyroid carcinomas. Ifdiagnosed at an early stage, thyroid cancer is a well manageable diseasewith a 5-year survival rate of 97% among all patients. Survival rate ispoorer (about 40%) among individuals that are diagnosed with a moreadvanced disease; i.e., individuals with large, invasive tumors and/ordistant metastases have a 5-year survival rate of about 40%. Forradioiodine-resistant metastatic disease, there is no effectivetreatment and the 10-year survival rate among these patients is lessthan 15%.

Although relatively rare (1% of all malignancies in the US), theincidence of thyroid cancer more than doubled between 1984 and 2004 inthe US. Between 1995 and 2004, thyroid cancer was the third fastestgrowing cancer diagnosis, behind only peritoneum, omentum, and mesenterycancers and “other” digestive cancers. Similarly, dramatic increases inthyroid cancer incidence have also been observed in Canada, Australia,Israel, and several European countries. Thus, there is a need for betterunderstanding of the molecular causes of thyroid cancer progression todevelop new diagnostic tools and better treatment options.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatcertain mutations in the promoter region of the telomerase reversetranscriptase (TERT) gene are prevalent in certain cancer—thyroid,bladder and glioblastoma, thus representing new diagnostic, prognosticand therapeutic targets.

More specifically, we explored mutations 1 295 228 C>T and 1 295 250 C>T(termed C228T and C250T respectively), corresponding to −124 C>T and−146 C>T from the translation start site in the promoter of thetelomerase reverse transcriptase (TERT) gene, in thyroid cancers bygenomic sequencing of a large number of primary tumor samples. We foundthe C228T mutation in 0 of 85 (0.0%) benign thyroid tumors, 30 of 257(11.7%) papillary thyroid cancers (PTC), 9 of 79 (11.4%) follicularthyroid cancers (FTC), 3 of 8 (37.5%) poorly differentiated thyroidcancers (PDTC), 23 of 54 (42.6%) anaplastic thyroid cancers (ATC), and 8of 12 (66.7%) thyroid cancer cell lines. The C250T mutation wasuncommon, but mutually exclusive with the C228T mutation, and the twomutations were collectively found in 11 of 79 (13.9%) FTC, 25 of 54(46.3%) ATC, and 11 of 12 (91.7%) thyroid cancer cell lines. Among PTCvariants, the C228T mutation was found in 4 of 13 (30.8%) tall-cell PTC(TCPTC), 23 of 187 (12.3%) conventional PTC, and 2 of 56 (3.6%)follicular variant PTC samples. No TERT mutation was found in 16medullary thyroid cancer samples. The C228T mutation was associated withthe BRAF V600E mutation in

PTC, being present in 19 of 104 (18.3%) BRAF mutation-positive PTC vs.11 of 153 (7.2%) the BRAF mutation-negative PTC samples (PZ0.0094).Conversely, BRAF mutation was found in 19 of 30 (63.3%) C228Tmutation-positive PTC vs. 85 of 227 (37.4%) C228T mutation-negative PTCsamples (PZ0.0094). Thus, for the first time, to our knowledge, wedemonstrate TERT promoter mutations in thyroid cancers that areparticularly prevalent in the aggressive thyroid cancers TCPTC, PDTC,ATC and BRAF mutation-positive PTC, revealing a novel genetic backgroundfor thyroid cancers. In addition, as further described herein, we foundhighly prevalent TERT promoter mutations in bladder cancer andglioblastoma.

Accordingly, in one aspect, the present invention provides methods oftreatment of subject having thyroid cancer. In one embodiment, a methodfor treating a subject having thyroid cancer comprises the steps of (a)obtaining a biological sample from the subject; (b) performing an assayon the sample obtained from the subject to identify a mutation at 1 295228 C>T (C228T) and 1 295 250 C>T (C250T), corresponding to −124 C>T and−146 C>T from the translation start site in the promoter of thetelomerase reverse transcriptase (TERT) gene; (c) identifying thesubject as having or likely to develop aggressive thyroid cancer if theC228T and/or C250T mutation is identified; and (d) treating the subjectwith one or more treatment modalities appropriate for a subject havingor likely to develop aggressive thyroid cancer. In a specificembodiment, the assay of step (b) comprises sequencing of the TERTpromoter region comprising −124 and −146 from the translation start sitein the promoter of TERT.

In another specific embodiment, the assay of step (b) comprises thesteps of (i) extracting DNA from the biological sample; (ii) contactingthe DNA with a primer that specifically hybridizes to the TERT gene;(iii) amplifying by polymerase chain reaction (PCR) a region of the TERTgene that comprises −124 and −146 from the translation start site in thepromoter of TERT; and (iv) sequencing the amplification product toidentify the presence of the C228T and/or C250T mutation. In certainembodiments, the primer comprises SEQ ID NO:2 and/or SEQ ID NO:3.

In particular embodiments, the treatment modality for aggressive thyroidcancer comprises one or more of thyroidectomy, hemithyroidectomy,radioactive iodine therapy, and combinations thereof. In a furtherembodiment, the treatment modality comprises administering to thesubject a TERT inhibitor.

In another aspect, the present invention provides methods of identifyinga subject as having or likely to develop aggressive thyroid cancer, andtreatment thereof. In one embodiment, a method for identifying a subjectas having or likely to develop aggressive thyroid cancer comprises thesteps of (a) obtaining a biological sample from the subject; (b)performing an assay on the sample obtained from the subject to identifya mutation at 1 295 228 C>T (C228T) and 1 295 250 C>T (C250T),corresponding to −124 C>T and −146 C>T from the translation start sitein the promoter of the telomerase reverse transcriptase (TERT) gene; and(c) identifying the subject as having or likely to develop aggressivethyroid cancer if the C228T and/or C250T mutation is identified. In aspecific embodiment, the assay of step (b) comprises sequencing of theTERT promoter region comprising −124 and −146 from the translation startsite in the promoter of TERT.

In another specific embodiment, the assay of step (b) comprises thesteps of (i) extracting DNA from the biological sample; (ii) contactingthe DNA with a primer that specifically hybridizes to the TERT gene;(iii) amplifying by polymerase chain reaction (PCR) a region of the TERTgene that comprises −124 and −146 from the translation start site in thepromoter of TERT; and (iv) sequencing the amplification product toidentify the presence of the C228T and/or C250T mutation. In certainembodiments, the primer comprises SEQ ID NO:2 and/or SEQ ID NO:3.

In certain embodiments, the method further comprises the step ofadministering a treatment modality appropriate for a subject having orlikely to develop aggressive thyroid cancer. In particular embodiments,the treatment modality for aggressive thyroid cancer comprisesthyroidectomy, hemithyroidectomy, radioactive iodine therapy, andcombinations thereof. In a further embodiment, the treatment modalitycomprises administering to the subject a TERT inhibitor.

Accordingly, in another aspect, the present invention provides methodsof treatment of subject having bladder cancer. In one embodiment, amethod for treating a subject having bladder cancer comprises the stepsof (a) obtaining a biological sample from the subject; (b) performing anassay on the sample obtained from the subject to identify a mutation at1 295 228 C>T (C228T) and 1 295 250 C>T (C250T), corresponding to −124C>T and −146 C>T from the translation start site in the promoter of thetelomerase reverse transcriptase (TERT) gene; (c) identifying thesubject as having or likely to develop bladder cancer if the C228Tand/or C250T mutation is identified; and (d) treating the subject withone or more treatment modalities appropriate for a subject having orlikely to develop bladder. In certain embodiments, the biological sampleis a urine sample. In a specific embodiment, the assay of step (b)comprises sequencing of the TERT promoter region comprising −124 and−146 from the translation start site in the promoter of TERT. In anotherspecific embodiment, the assay of step (b) comprises the steps of (i)extracting DNA from the biological sample; (ii) contacting the DNA witha primer that specifically hybridizes to the TERT gene; (iii) amplifyingby polymerase chain reaction (PCR) a region of the TERT gene thatcomprises −124 and −146 from the translation start site in the promoterof TERT; and (iv) sequencing the amplification product to identify thepresence of the C228T and/or C250T mutation. In certain embodiments, theprimer comprises SEQ ID NO:2 and/or SEQ ID NO:3.

In another aspect, the present invention provides methods of identifyinga subject as having or likely to develop bladder cancer, and treatmentthereof. In one embodiment, a method for identifying a subject as havingor likely to develop bladder cancer comprises the steps of (a) obtaininga biological sample from the subject; (b) performing an assay on thesample obtained from the subject to identify a mutation at 1 295 228 C>T(C228T) and 1 295 250 C>T (C250T), corresponding to −124 C>T and −146C>T from the translation start site in the promoter of the telomerasereverse transcriptase (TERT) gene; and (c) identifying the subject ashaving or likely to develop bladder cancer if the C228T and/or C250Tmutation is identified. In a specific embodiment, the assay of step (b)comprises sequencing of the TERT promoter region comprising −124 and−146 from the translation start site in the promoter of TERT.

In another specific embodiment, the assay of step (b) comprises thesteps of (i) extracting DNA from the biological sample; (ii) contactingthe DNA with a primer that specifically hybridizes to the TERT gene;(iii) amplifying by polymerase chain reaction (PCR) a region of the TERTgene that comprises −124 and −146 from the translation start site in thepromoter of TERT; and (iv) sequencing the amplification product toidentify the presence of the C228T and/or C250T mutation. In certainembodiments, the primer comprises SEQ ID NO:2 and/or SEQ ID NO:3. Inparticular embodiments, the biological sample is a urine sample.

In particular embodiments, the treatment modality for bladder cancercomprises one or more of surgery including, but not limited to,transurethral resection (with fulguration), radical cystectomy, partialcystectomy, and urinary diversion; radiation therapy, chemotherapy(e.g., intravesical); and biologic therapy such as BCG (bacillusCalmette Guerin). In a further embodiment, the treatment modalitycomprises administering to the subject a TERT inhibitor.

In a further aspect, the present invention provides methods of treatmentof subject having glioblastoma. In one embodiment, a method for treatinga subject having glioblastoma comprises the steps of (a) obtaining abiological sample from the subject; (b) performing an assay on thesample obtained from the subject to identify a mutation at 1 295 228 C>T(C228T) and 1 295 250 C>T (C250T), corresponding to −124 C>T and −146C>T from the translation start site in the promoter of the telomerasereverse transcriptase (TERT) gene; (c) identifying the subject as havingor likely to develop glioblastoma if the C228T and/or C250T mutation isidentified; and (d) treating the subject with one or more treatmentmodalities appropriate for a subject having or likely to developbladder. In a specific embodiment, the assay of step (b) comprisessequencing of the TERT promoter region comprising −124 and −146 from thetranslation start site in the promoter of TERT. In another specificembodiment, the assay of step (b) comprises the steps of (i) extractingDNA from the biological sample; (ii) contacting the DNA with a primerthat specifically hybridizes to the TERT gene; (iii) amplifying bypolymerase chain reaction (PCR) a region of the TERT gene that comprises−124 and −146 from the translation start site in the promoter of TERT;and (iv) sequencing the amplification product to identify the presenceof the C228T and/or C250T mutation. In certain embodiments, the primercomprises SEQ ID NO:2 and/or SEQ ID NO:3.

In yet another aspect, the present invention provides methods ofidentifying a subject as having or likely to develop glioblastoma, andtreatment thereof. In one embodiment, a method for identifying a subjectas having or likely to develop glioblastoma comprises the steps of (a)obtaining a biological sample from the subject; (b) performing an assayon the sample obtained from the subject to identify a mutation at 1 295228 C>T (C228T) and 1 295 250 C>T (C250T), corresponding to −124 C>T and−146 C>T from the translation start site in the promoter of thetelomerase reverse transcriptase (TERT) gene; and (c) identifying thesubject as having or likely to develop glioblastoma if the C228T and/orC250T mutation is identified. In a specific embodiment, the assay ofstep (b) comprises sequencing of the TERT promoter region comprising−124 and −146 from the translation start site in the promoter of TERT.

In another specific embodiment, the assay of step (b) comprises thesteps of (i) extracting DNA from the biological sample; (ii) contactingthe DNA with a primer that specifically hybridizes to the TERT gene;(iii) amplifying by polymerase chain reaction (PCR) a region of the TERTgene that comprises −124 and −146 from the translation start site in thepromoter of TERT; and (iv) sequencing the amplification product toidentify the presence of the C228T and/or C250T mutation. In certainembodiments, the primer comprises SEQ ID NO:2 and/or SEQ ID NO:3. Inparticular embodiments, the biological sample is a urine sample. Inparticular embodiments, the treatment modality for brain cancerincluding glioblastoma comprises one or more of surgery, radiationtherapy (e.g., 3-dimensional radiation therapy, intensity-modulatedradiation therapy, sterotactic radiation therapy, and proton beamradiation therapy); chemotherapy (e.g., intrathecal); and biologictherapy (e.g., tyrosine kinase inhibitor therapy, vascular endothelialgrowth factor (VEGF) therapy, dendritic call vaccine therapy, and genetherapy). In a further embodiment, the treatment modality comprisesadministering to the subject a TERT inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B. Sequencing of the human TERT promoter electropherograms.Representative electropherograms of the genomic DAN sequencing of thehuman TERT promoter for the two indicated mutations are shown. FIG. 1A:The sense DNA strand obtained using the sense primer for sequencing,displaying TERT promoter mutations C228T and C250T in various thyroidcancer cell lines and thyroid cancer samples. FIG. 1B: The antisense DNAstrand obtained using the antisense primer for sequencing, displayingTERT promoter mutations G228A and G250A in various thyroid cancer celllines and thyroid cancer samples. FIG. 1A and 1B: WRO cell line is usedto show the wild-type human TERT promoter. PTC, papillary thyroidcancer; FTC, follicular thyroid cancer; ATC, anaplastic thyroid cancer;PDTC, poorly differentiated thyroid cancer.

FIG. 2. TERT promoter mutations in bladder cancer and glioblastoma.Shown are representative electropherograms of the wild-type TERTpromoter and two TERT promoter mutations as indicated for bladder cancerand glioblastoma. The upper portion of the figure showselectropherograms of the sense sequences of the wild-type DNA and thenucleotide changes of the two mutations in the two cancers. The lowerportion of the figure shows the electropherograms of the antisensesequences of the wild-type DNA and the nucleotide changes of the twomutations in the two cancers.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

I. Definitions

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. Unless specifically stated or obvious fromcontext, as used herein, the term “about” is understood as within arange of normal tolerance in the art, for example within 2 standarddeviations of the mean. About can be understood as within 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the statedvalue. Unless otherwise clear from context, all numerical valuesprovided herein can be modified by the term “about.”

An “agonist” is a type of modulator and refers to an agent that binds atarget and can activate one or more functions of the target. Forexample, an agonist of a protein can bind the protein and activate theprotein in the absence of its natural or cognate ligand.

As used herein, an “antagonist” is a type of modulator and is usedinterchangeably with the term “inhibitor.” In certain non-limitingembodiments, the term refers to an agent that binds a target (e.g., aprotein) and can inhibit a one or more functions of the target. Forexample, an antagonist of an enzymatic protein can bind the protein andinhibit the enzymatic activity of the protein.

As used herein, the term “antibody” is used in reference to anyimmunoglobulin molecule that reacts with a specific antigen. It isintended that the term encompass any immunoglobulin (e.g., IgG, IgM,IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents,non-human primates, caprines, bovines, equines, ovines, etc.). Specifictypes/examples of antibodies include polyclonal, monoclonal, humanized,chimeric, human, or otherwise-human-suitable antibodies. “Antibodies”also includes any fragment or derivative of any of the herein describedantibodies. In specific embodiments, antibodies may be raised againstTERT and used as TERT modulators.

As used herein, the term “effective,” means adequate to accomplish adesired, expected, or intended result. More particularly, a“therapeutically effective amount” as provided herein refers to anamount of a TERT modulator of the present invention, either alone or incombination with another therapeutic agent, necessary to provide thedesired therapeutic effect, e.g., an amount that is effective toprevent, alleviate, or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. In a specific embodiment, theterm “therapeutically effective amount” as provided herein refers to anamount of a TERT modulator, necessary to provide the desired therapeuticeffect, e.g., an amount that is effective to prevent, alleviate, orameliorate symptoms of disease or prolong the survival of the subjectbeing treated. In a particular embodiment, the disease or condition iscancer. In a more specific embodiments, the cancer is thyroid cancer. Aswould be appreciated by one of ordinary skill in the art, the exactamount required will vary from subject to subject, depending on age,general condition of the subject, the severity of the condition beingtreated, the particular compound and/or composition administered, andthe like. An appropriate “therapeutically effective amount” in anyindividual case can be determined by one of ordinary skill in the art byreference to the pertinent texts and literature and/or by using routineexperimentation.

By “high stringency conditions” is meant conditions that allowhybridization comparable with that resulting from the use of a DNA probeof at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well-known by thoseskilled in the art of molecular biology. (See, for example, F. Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1998).

The term “inhibitor” is a type of modulator and is used interchangeablywith the term “antagonist.” The term “inhibitor” includes any type ofmolecule or agent that directly or indirectly inhibits the expression oractivity of a target gene or protein. An inhibitor can be any type ofcompound, such as a small molecule, antibody or antisense compound. Incertain embodiments, the target gene or protein is TERT. The term alsoincludes agents that have activity in addition to TERT inhibitoryactivity.

As used herein, the term “modulate” indicates the ability to control orinfluence directly or indirectly, and by way of non-limiting examples,can alternatively mean inhibit or stimulate, agonize or antagonize,hinder or promote, and strengthen or weaken. Thus, the term “TERTmodulator” refers to an agent that modulates the expressions and/oractivity of TERT. Modulators may be organic or inorganic, small to largemolecular weight individual compounds, mixtures and combinatoriallibraries of inhibitors, agonists, antagonists, and biopolymers such aspeptides, nucleic acids, or oligonucleotides. A modulator may be anatural product or a naturally-occurring small molecule organiccompound. In particular, a modulator may be a carbohydrate;monosaccharide; oligosaccharide; polysaccharide; amino acid; peptide;oligopeptide; polypeptide; protein; receptor; nucleic acid; nucleoside;nucleotide; oligonucleotide; polynucleotide including DNA and DNAfragments, RNA and RNA fragments and the like; lipid; retinoid; steroid;glycopeptides; glycoprotein; proteoglycan and the like; and syntheticanalogues or derivatives thereof, including peptidomimetics, smallmolecule organic compounds and the like, and mixtures thereof. Amodulator identified according to the invention is preferably useful inthe treatment of a disease disclosed herein.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof.

Optional” or “optionally” means that the subsequently described event orcircumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The terms “patient,” “individual,” or “subject” are used interchangeablyherein, and refer to a mammal, particularly, a human. The patient mayhave a mild, intermediate or severe disease or condition. The patientmay be treatment naïve, responding to any form of treatment, orrefractory. The patient may be an individual in need of treatment or inneed of diagnosis based on particular symptoms or family history. Insome cases, the terms may refer to treatment in experimental animals, inveterinary application, and in the development of animal models fordisease, including, but not limited to, rodents including mice, rats,and hamsters; and primates. In particular, the term also includesmammals diagnosed with a TERT mediated disease, disorder or condition.By “normal subject” is meant an individual who does not have cancer aswell as an individual who has increased susceptibility for developing acancer.

“Polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules. In addition, asused herein, the term “polypeptide” refers to amino acids joined to eachother by peptide bonds or modified peptide bonds, e.g., peptideisosteres, etc., and may contain modified amino acids other than the 20gene-encoded amino acids. The polypeptides can be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Modificationscan occur anywhere in the polypeptide, including the peptide backbone,the amino acid side-chains and the amino or carboxyl termini. The sametype of modification can be present in the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide can havemany types of modifications. Modifications include, without limitation,acetylation, acylation, ADP-ribosylation, amidation, covalentcross-linking or cyclization, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphytidylinositol, disulfidebond formation, demethylation, formation of cysteine or pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pergylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. SeeProteins—Structure and Molecular Properties 2nd Ed., T. E. Creighton,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

By “probe,” “primer,” or oligonucleotide is meant a single-stranded DNAor RNA molecule of defined sequence that can base-pair to a second DNAor RNA molecule that contains a complementary sequence (the “target”).The stability of the resulting hybrid depends upon the extent of thebase-pairing that occurs. The extent of base-pairing is affected byparameters such as the degree of complementarity between the probe andtarget molecules and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as temperature, salt concentration, and theconcentration of organic molecules such as formamide, and is determinedby methods known to one skilled in the art. Probes or primers specificfor TERT nucleic acids (for example, genes and/or mRNAs) have at least80%-90% sequence complementarity, preferably at least 91%-95% sequencecomplementarity, more preferably at least 96%-99% sequencecomplementarity, and most preferably 100% sequence complementarity tothe region of the TERT nucleic acid to which they hybridize. Probes,primers, and oligonucleotides may be detectably-labeled, eitherradioactively, or non-radioactively, by methods well-known to thoseskilled in the art. Probes, primers, and oligonucleotides are used formethods involving nucleic acid hybridization, such as: nucleic acidsequencing, reverse transcription and/or nucleic acid amplification bythe polymerase chain reaction, single stranded conformationalpolymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP)analysis, Southern hybridization, Northern hybridization, in situhybridization, electrophoretic mobility shift assay (EMSA).

The terms “sample,” “patient sample,” “biological sample,” and the like,encompass a variety of sample types obtained from a patient, individual,or subject and can be used in a diagnostic or monitoring assay. Thepatient sample may be obtained from a healthy subject or a patienthaving symptoms associated with prostate cancer. Moreover, a sampleobtained from a patient can be divided and only a portion may be usedfor diagnosis. Further, the sample, or a portion thereof, can be storedunder conditions to maintain sample for later analysis. The definitionspecifically encompasses blood and other liquid samples of biologicalorigin (including, but not limited to, peripheral blood, serum, plasma,cord blood, amniotic fluid, cerebrospinal fluid, urine, saliva, stooland synovial fluid), solid tissue samples such as a biopsy specimen ortissue cultures or cells derived therefrom and the progeny thereof. Incertain embodiments, a sample comprises blood. In other embodiments, asample comprises serum. In a specific embodiment, a sample comprisesplasma. In yet another embodiment, a semen sample is used. In a furtherembodiment, a stool sample is used. In particular embodiments, TERTpromoter mutations described here can be tested on tumor tissues,including surgical tissues, needle biopsy tissues (e.g., thyroid noduleneedle biopsy specimens), body fluids (e.g., needle biopsy washings,cerebral spinal fluids, urine, etc.) for the diagnosis, prognosis andtreatment guidance and treatments of cancer, such as thyroid cancer,bladder cancer, brain tumor/glioblastoma, and other cancers.

In certain embodiments, and in particular for the identification andtreatment of bladder cancer, a sample comprises urine. Indeed, TERTmutations can be detected in urine as molecular markers for thediagnosis, prognostication and treatment of bladder cancer. See Hurst etal., 65 European Urology 367-69 (2014) (“Comprehensive Mutation Analysisof the TERT Promoter in Bladder Cancer and Detection of Mutations inVoided Urine”); and Rochakonda et al., 110(43) Proc. Natl. Acad. Sci.USA 17426-17431 (October 2013) (“TERT Promoter Mutations in BladderCancer Affect Patient Survival and Disease Recurrence ThroughModification by a Common Polymorphism”).

The definition of “sample” also includes samples that have beenmanipulated in any way after their procurement, such as bycentrifugation, filtration, precipitation, dialysis, chromatography,treatment with reagents, washed, or enriched for certain cellpopulations. The terms further encompass a clinical sample, and alsoinclude cells in culture, cell supernatants, tissue samples, organs, andthe like. Samples may also comprise fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks, such as blocks prepared from clinicalor pathological biopsies, prepared for pathological analysis or study byimmunohistochemistry.

The terms “specifically binds to,” “specific for,” and relatedgrammatical variants refer to that binding which occurs between suchpaired species as antibody/antigen, enzyme/substrate, receptor/agonist,and lectin/carbohydrate which may be mediated by covalent ornon-covalent interactions or a combination of covalent and non-covalentinteractions. When the interaction of the two species produces anon-covalently bound complex, the binding which occurs is typicallyelectrostatic, hydrogen-bonding, or the result of lipophilicinteractions. Accordingly, “specific binding” occurs between a pairedspecies where there is interaction between the two which produces abound complex having the characteristics of an antibody/antigen orenzyme/substrate interaction. In particular, the specific binding ischaracterized by the binding of one member of a pair to a particularspecies and to no other species within the family of compounds to whichthe corresponding member of the binding member belongs. Thus, forexample, an antibody typically binds to a single epitope and to no otherepitope within the family of proteins. In some embodiments, specificbinding between an antigen and an antibody will have a binding affinityof at least 10⁻⁶ M. In other embodiments, the antigen and antibody willbind with affinities of at least 10⁻⁷ M, 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, or 10⁻¹² M.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a TERT nucleic acid) under high stringency conditions, and doesnot substantially base pair with other nucleic acids.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a subject, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, e.g., causing regression of the disease,e.g., to completely or partially remove symptoms of the disease. In aspecific embodiment, the disease or condition is cancer including, butnot limited to, thyroid, bladder and glioblastoma.

The terms “TERT-related disease, disorder or condition” or“TERT-mediated disease, disorder or condition,” and the like meandiseases, disorders or conditions associated with aberrant TERTactivity. In a specific embodiment, the disease or condition is cancer .In general, the term refers to any abnormal state that involves TERTactivity. The abnormal state can be due, for example, to a geneticdefect.

II. TERT Promoter Mutations as Biomarkers

The present inventors have discovered that certain mutations in thepromoter region of TERT are associated with cancers including, but notlimited to, thyroid cancer, bladder cancer and glioblastoma. Thyroidcancer can include follicular thyroid cancer (FTC), papillary thyroidcancer (PTC), conventional PTC, follicular variant PTC (FVPTC),tall-cell PTC (TCPTC).

Thus, in certain embodiments, the TERT promoter mutations can thus beused to identify individuals having or at risk of developing cancer. Infurther embodiments, the TERT promoter mutations can be used to identifyindividuals at risk for having or developing aggressive thyroid cancersuch as TCPTC, PDTC, ATC, and BRAF-mutation-positive PTC. The mutationscan be identified in subjects who have or have not been diagnosed withcancer.

In certain embodiments, DNA can be isolated from a biological sampletaken from a subject. DNA can be extracted and purified from biologicalsamples using any suitable technique. A number of techniques for DNAextraction and/or purification are known in the art, and several arecommercially available (e.g., ChargeSwitch®, MELT™ total nucleic acidisolation system, MagMAX™ FFPE total nucleic acid isolation kit, MagMAX™total nucleic acid isolation kit, QIAamp DNA kit, Omni-Pure™ genomic DNApurification system, WaterMaster™ DNA purification kit). Reagents suchas DNAzoI® and TR1 Reagent® can also be used to extract and/or purifyDNA. DNA can be further purified using Proteinase K and/or RNAse.

In further embodiments, primer/probes can be used to amplify a region ofthe TERT gene that comprises the promoter. More specifically,primers/probes are capable of amplifying the promoter region at 1 295228 C>T and 1 295 250 C>T (termed C228T and C250T respectively),corresponding to −124 C>T and −146 C>T from the translation start sitein the promoter of the telomerase reverse transcriptase (TERT) gene. Inone embodiment, a primer comprises the nucleic acid sequence shown inSEQ ID NO:2. In another embodiment, a primer comprises the nucleic acidsequence shown in SEQ ID NO:3. A primer set can comprise the nucleicacid sequences shown in SEQ ID NO:2 and SEQ ID NO:3.

In particular embodiments, a primer is contacted with isolated DNA fromthe subject under conditions such that the primer specificallyhybridizes with the TERT gene. The primer and DNA thus form a primer:DNAcomplex. In certain embodiments, the hybridization conditions are suchthat the formation of the primer:DNA complex is the detection stepitself, i.e., the complex forms only if the mutation (C228T and/orC250T) is present. In other embodiments, the primer:DNA complex isamplified using polymerase chain reaction, the presence (or not) of themutation is detected. In certain embodiments, the mutations are detectedby sequencing.

As described herein, in certain embodiments, the primers can used tosupport DNA amplification reactions. Typically the primers will becapable of being extended in a sequence specific manner. Extension of aprimer in a sequence specific manner includes any methods wherein thesequence or composition of the nucleic acid molecule to which the primeris hybridized or otherwise associated directs or influences thecomposition or sequence of the product produced by the extension of theprimer. Extension of the primer in a sequence specific manner thereforeincludes, but is not limited to, PCR, DNA sequencing, DNA extension, DNApolymerization, RNA transcription, or reverse transcription. Techniquesand conditions that amplify the primer in a sequence specific manner arepreferred. In certain embodiments the primers are used for the DNAamplification reactions, such as PCR or direct sequencing. It isunderstood that in certain embodiments the primers can also be extendedusing non-enzymatic techniques, where for example, the nucleotides oroligonucleotides used to extend the primer are modified such that theywill chemically react to extend the primer in a sequence specificmanner. Typically the disclosed primers hybridize with thepolynucleotide sequences disclosed herein or region of thepolynucleotide sequences disclosed herein or they hybridize with thecomplement of the polynucleotide sequences disclosed herein orcomplement of a region of the polynucleotide sequences disclosed herein.

The size of the primers or probes for interaction with thepolynucleotide sequences disclosed herein in certain embodiments can beany size that supports the desired enzymatic manipulation of the primer,such as DNA amplification or the simple hybridization of the probe orprimer. A typical primer or probe would be at least 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,3500, or 4000 nucleotides long or any length in-between.

The probes or primers of the present invention can be prepared byconventional techniques well-known to those skilled in the art. Forexample, the probes can be prepared using solid-phase synthesis usingcommercially available equipment. Modified oligonucleotides can also bereadily prepared by similar methods. The probes can also be synthesizeddirectly on a solid support according to methods standard in the art.This method of synthesizing polynucleotides is particularly useful whenthe polynucleotide probes are part of a nucleic acid array.

The present invention therefore also provides predictive, diagnostic,and prognostic kits comprising degenerate primers to amplify a targetnucleic acid in the TERT gene and instructions comprising amplificationprotocol and analysis of the results. The kit may alternatively alsocomprise buffers, enzymes, and containers for performing theamplification and analysis of the amplification products. The kit mayalso be a component of a screening, diagnostic or prognostic kitcomprising other tools such as DNA microarrays. In some embodiments, thekit also provides one or more control templates, such as nucleic acidsisolated from normal tissue sample, and/or a series of samplesrepresenting different variances in the TERT gene.

In one embodiment, the kit provides at least one primer capable ofamplifying a different region of the TERT gene. The kit may compriseadditional primers for the analysis of expression of several genevariances in a biological sample in one reaction or several parallelreactions. Primers in the kits may be labeled, for example fluorescentlylabeled, to facilitate detection of the amplification products andconsequent analysis of the nucleic acid variances.

In one embodiment, more than one mutation/variance can be detected inone analysis. A combination kit will therefore comprise of primerscapable of amplifying different segments of the TERT gene. A kit mayalso comprise primers capable of amplifying segments of another gene(s)including BRAF. The primers may be differentially labeled, for example,using different fluorescent labels, so as to differentiate between thevariances. The primers contained within the kit may include primersselected from complementary sequences to the coding sequence of TERT.

In certain embodiments, a patient can be diagnosed or identified byadding a biological sample (e.g., blood or blood serum) obtained fromthe patient to the kit and detecting the TERT promoter mutations(s), forexample, by a method which comprises the steps of: (i) collecting bloodor blood serum from the patient; (ii) separating DNA from the patient'sblood; (iii) adding the DNA from patient to a diagnostic kit; and, (iv)detecting (or not) the TERT promoter mutation(s). In this exemplarymethod, primers are brought into contact with the patient's DNA. Theformation of the primer:DNA complex can, for example, be PCR amplifiedand, in some embodiments, sequenced to detect (or not) the

TERT promoter mutation. In other kit and diagnostic embodiments, bloodor blood serum need not be collected from the patient (i.e., it isalready collected). Moreover, in other embodiments, the sample maycomprise a tissue sample, urine or a clinical sample.

III. TERT Modulators

In certain embodiments, the TERT modulator is selected from the groupconsisting of a small molecule, a polypeptide, a nucleic acid molecule,a peptidomimetic, or a combination thereof. In a specific embodiment,the agent can be a polypeptide. The polypeptide can, for example,comprise an antibody. In another embodiment, the agent can be a nucleicacid molecule. The nucleic acid molecule can, for example, be a TERTinhibitory nucleic acid molecule. The TERT inhibitory nucleic acidmolecule can comprise a short interfering RNA (siRNA) molecule, amicroRNA (miRNA) molecule, or an antisense molecule.

A. Antibodies to TERT

The term antibody is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. The term can also refer to a humanantibody and/or a humanized antibody. Examples of techniques for humanmonoclonal antibody production include those described by Cole et al.(Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985))and by Boerner et al. (J. Immunol. 147(1):86-95 (1991)). Humanantibodies (and fragments thereof) can also be produced using phagedisplay libraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991);Marks et al., J. Mol. Biol. 222:581 (1991)). The disclosed humanantibodies can also be obtained from transgenic animals. For example,transgenic mutant mice that are capable of producing a full repertoireof human antibodies, in response to immunization, have been described(see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-5(1993); Jakobovits et al., Nature 362:255-8 (1993); Bruggermann et al.,Year in Immunol. 7:33 (1993)).

Various procedures known in the art may be used for the production ofantibodies to TERT or any subunit thereof, or a fragment, derivative,homolog or analog of the protein. Antibodies of the present inventioninclude, but are not limited to, synthetic antibodies, polyclonalantibodies, monoclonal antibodies, recombinantly produced antibodies,intrabodies, multispecific antibodies (including bi-specificantibodies), human antibodies, humanized antibodies, chimericantibodies, synthetic antibodies, single-chain Fvs (scFv) (includingbi-specific scFvs), single chain antibodies Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Inparticular, antibodies of the present invention include immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, e.g., molecules that contain an antigen binding site thatimmunospecifically binds to an antigen (e.g., one or morecomplementarity determining regions (CDRs) of an antibody).

Another embodiment for the preparation of antibodies according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsin rational design is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used to engineer secondgeneration molecules having many of the natural properties of thetargeting antibodies disclosed herein, but with altered and evenimproved characteristics. More specifically, under this rational designapproach, peptide mapping may be used to determine “active” antigenrecognition residues, and along with molecular modeling and moleculardynamics trajectory analysis, peptide mimic of the antibodies containingantigen contact residues from multiple CDRs may be prepared.

In some embodiments, an antibody specifically binds an epitope of theTERT protein. It is to be understood that the peptide regions may notnecessarily precisely map one epitope, but may also contain a TERTsequence that is not immunogenic. Methods of predicting other potentialepitopes to which an immunoglobulin of the invention can bind arewell-known to those of skill in the art and include, without limitation,Kyte-Doolittle Analysis (Kyte, J. and Dolittle, R. F., 157 J. MOL. BIOL.105-32 (1982)); Hopp and Woods Analysis (Hopp, T. P. and Woods, K. R.,78 PROC. NATL. ACAD. SCI. USA 3824-28 (1981); Hopp, T. J. and Woods, K.R., 20 MOL. IMMUNOL. 483-89 (1983); Hopp, T. J., 88 J. IMMUNOL. METHODS1-18 (1986)); Jameson-Wolf Analysis (Jameson, B. A. and Wolf, H., 4COMPUT. APPL. BIOSCI. 181-86 (1988)); and Emini Analysis (Emini et al.,140 VIROLOGY 13-20 (1985)).

Amino acid sequence variants of the antibodies of the present inventionmay be prepared by introducing appropriate nucleotide changes into thepolynucleotide that encodes the antibody or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletions, insertions, andsubstitutions may be made to arrive at the final construct.

Amino acid sequence insertions include amino-terminal and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the antibody fused to a cytotoxic polypeptide.Other insertional variants of the antibody molecule include the fusionto the N- or C-terminus of the antibody of a polypeptide that increasesthe serum half-life of the antibody.

Another type of antibody variant is an amino acid substitution variant.These variants have at least one amino acid residue in the antibodymolecule replaced by a different residue. For example, the sites ofgreatest interest for substitutional mutagenesis of antibodies includethe hypervariable regions, but framework region (FR) alterations arealso contemplated.

A useful method for the identification of certain residues or regions ofthe TERT antibodies that are preferred locations for substitution, i.e.,mutagenesis, is alanine scanning mutagenesis. See Cunningham & Wells,244 SCIENCE 1081-85 (1989). Briefly, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with antigen. The amino acid locations demonstratingfunctional sensitivity to the substitutions are refined by introducingfurther or other variants at, or for, the sites of substitution. Thus,while the site for introducing an amino acid sequence variation ispredetermined, the nature of the mutation per se need not bepredetermined. For example, to analyze the performance of a mutation ata given site, alanine scanning or random mutagenesis may be conducted atthe target codon or region and the expressed antibody variants screenedfor the desired activity.

Substantial modifications in the biological properties of the antibodycan be accomplished by selecting substitutions that differ significantlyin their effect on, maintaining (i) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (ii) the charge or hydrophobicity of the moleculeat the target site, or (iii) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gln, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Conservative substitutions involveexchanging of amino acids within the same class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is animmunoglobulin fragment such as an Fv fragment.

Another type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody. Generally, theresulting variant(s), i.e., functional equivalents as defined above,selected for further development will have improved biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsis by affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g., 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g., binding affinity) as herein disclosed.

In order to identify candidate hypervariable region sites formodification, alanine-scanning mutagenesis may be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antibody-antigen complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

It may be desirable to modify the antibodies of the present invention,i.e., create functional equivalents, with respect to effector function,e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity(ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody.This may be achieved by introducing one or more amino acid substitutionsin an Fc region of an antibody. Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). Caron et al., 176 J. EXP MED. 1191-95(1992); Shopes, 148 J. IMMUNOL. 2918-22 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., 53 CANCERRESEARCH 2560-65 (1993). Alternatively, an antibody can be engineeredwhich has dual Fc regions and may thereby have enhanced complement lysisand ADCC capabilities. Stevenson et al., 3 ANTI-CANCER DRUG DESIGN219-30 (1989).

To increase the serum half-life of an antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially animmunoglobulin fragment) as described in, for example, U.S. Pat. No.5,739,277. As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1,IgG2, IgG3, or IgG4) that is responsible for increasing the in vivoserum half-life of the IgG molecule.

Polynucleotide molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the anti-TERT antibodies of the presentinvention.

B. Small Molecule Modulators of TERT

In other embodiments, a TERT modulator is a small molecule. In aspecific embodiment, the TERT modulator is the antagonist BIBR1532(2-[(E)-3-naphthen-2-yl but-2-enoylamino]benzoic acid). See Ward &Autexier, Mol. Pharmacol. 68:779-786, 2005; also J. Biol. Chem.277(18):15566-72, 2002). TERT modulator antagonists can also includeTMPyP4 (tetra-(N-methyl-4-pyridyl)porphyrin), telomerase inhibitor IX(MST312), MnTMPyp pentachloride, BPPA, β-Rubromycin, Trichostatin A,Costunolide, Doxorubicin, Suramin Sodum, and (−)-Epigallocatchin Gallate(and other catechins). The term “small molecule organic compounds”refers to organic compounds generally having a molecular weight lessthan about 5000, 4000, 3000, 2000, 1000, 800, 600, 500, 250 or 100Daltons, preferably less than about 500 Daltons. A small moleculeorganic compound may be prepared by synthetic organic techniques, suchas by combinatorial chemistry techniques, or it may be anaturally-occurring small molecule organic compound.

Compound libraries may be screened for TERT modulators. A compoundlibrary is a mixture or collection of one or more putative modulatorsgenerated or obtained in any manner. Any type of molecule that iscapable of interacting, binding or has affinity for TERT may be presentin the compound library. For example, compound libraries screened usingthis invention may contain naturally-occurring molecules, such ascarbohydrates, monosaccharides, oligosaccharides, polysaccharides, aminoacids, peptides, oligopeptides, polypeptides, proteins, receptors,nucleic acids, nucleosides, nucleotides, oligonucleotides,polynucleotides, including DNA and DNA fragments, RNA and RNA fragmentsand the like, lipids, retinoids, steroids, glycopeptides, glycoproteins,proteoglycans and the like; or analogs or derivatives ofnaturally-occurring molecules, such as peptidomimetics and the like; andnon-naturally occurring molecules, such as “small molecule” organiccompounds generated, for example, using combinatorial chemistrytechniques; and mixtures thereof

A library typically contains more than one putative modulator or member,i.e., a plurality of members or putative modulators. In certainembodiments, a compound library may comprise less than about 50,000,25,000, 20,000, 15,000, 10000, 5000, 1000, 500 or 100 putativemodulators, in particular from about 5 to about 100, 5 to about 200, 5to about 300, 5 to about 400, 5 to about 500, 10 to about 100, 10 toabout 200, 10 to about 300, 10 to about 400, 10 to about 500, 10 toabout 1000, 20 to about 100, 20 to about 200, 20 to about 300, 20 toabout 400, 20 to about 500, 20 to about 1000, 50 to about 100, 50 toabout 200, 50 to about 300, 50 to about 400, 50 to about 500, 50 toabout 1000, 100 to about 200, 100 to about 300, 100 to about 400, 100 toabout 500, 100 to about 1000, 200 to about 300, 200 to about 400, 200 toabout 500, 200 to about 1000, 300 to about 500, 300 to about 1000, 300to 2000, 300 to 3000, 300 to 5000, 300 to 6000, 300 to 10,000, 500 toabout 1000, 500 to about 2000, 500 to about 3000, 500 to about 5000, 500to about 6000, or 500 to about 10,000 putative modulators. In particularembodiments, a compound library may comprise less than about 50,000,25,000, 20,000, 15,000, 10,000, 5,000, 1000, or 500 putative modulators.

A compound library may be prepared or obtained by any means including,but not limited to, combinatorial chemistry techniques, fermentationmethods, plant and cellular extraction procedures and the like. Alibrary may be obtained from synthetic or from natural sources such asfor example, microbial, plant, marine, viral and animal materials.Methods for making libraries are well-known in the art. See, forexample, E. R. Felder, Chimia 1994, 48, 512-541; Gallop et al., J. Med.Chem. 1994, 37, 1233-1251; R. A. Houghten, Trends Genet. 1993, 9,235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al., Nature1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden etal., Perspectives in Drug Discovery and Design 2, 269-282; Cwirla etal., Biochemistry 1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad.Sci. USA 1992, 89, 5381-5383; Gordon et al., J. Med. Chem. 1994, 37,1385-1401; Lebl et al., Biopolymers 1995, 37 177-198; and referencescited therein. Compound libraries may also be obtained from commercialsources including, for example, from Maybridge, ChemNavigator.com,Timtec Corporation, ChemBridge Corporation, A-Syntese-Biotech ApS,Akos-SC, G & J Research Chemicals Ltd., Life Chemicals, Interchim S.A.,and Spectrum Info. Ltd.

C. RNA Interference Compositions for Targeting TERT mRNAs

In one aspect of the present invention, the expression of TERT may beinhibited by the use of RNA interference techniques (RNAi). RNAi is aremarkably efficient process whereby double-stranded RNA (dsRNA) inducesthe sequence-specific degradation of homologous mRNA in animals andplant cells. See Hutvagner and Zamore, 12 CURR. OPIN. GENET. DEV. 225-32(2002); Hammond et al., 2 NATURE REV. GEN. 110-19 (2001); Sharp, 15GENES DEV. 485-90 (2001). RNAi can be triggered, for example, bynucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al.,10 MOL. CELL. 549-61 (2002); Elbashir et al., 411 Nature 494-98 (2001)),micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or otherdsRNAs which are expressed in-vivo using DNA templates with RNApolymerase III promoters. See, e.g., Zeng et al., 9 MOL. CELL. 1327-33(2002); Paddison et al., 16 GENES DEV. 948-58 (2002); Lee et al., 20NATURE BIOTECHNOL. 500-05 (2002); Paul et al., 20 NATURE BIOTECHNOL.505-08 (2002); Tuschl, 20 NATURE BIOTECHNOL. 440-48 (2002); Yu et al.,99(9) PROC. NATL. ACAD. SCI. USA, 6047-52 (2002); McManus et al., 8 RNA842-50 (2002); Sui et al., 99(6) PROC. NATL. ACAD. SCI. USA 5515-20(2002).

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Highly Prevelant TERT Promoter Mutations in Aggressive Thyroid Cancers

Telomerase, a ribonucleoprotein complex that maintains telomere lengthat the end of chromosomes, plays a key role in cellular immortality andtumorigenesis (Smekalova et al. 2012, Mocellin et al. 2013). Itscatalytic subunit is telomerase reverse transcriptase (TERT). Promotermutations in the TERT gene on chromosome 5 have recently been reportedin melanomas (Horn et al. 2013, Huang et al. 2013). Two TERT promotermutations, 1 295 228 C>T and 1 295 250 C>T (termed C228T and C250T hererespectively), are particularly common. They represent nucleotidechanges of −124 C>T and −146 C>T (where -1 is the base just upstream ofthe A of the ATG translation start site) respectively in the TERTpromoter. Both the mutations create an 11-base nucleotide stretch5′-CCCCTTCCGGG-3′ (SEQ ID NO:1), which contains a consensus bindingsite, GGAA (in reverse complement), for ETS transcription factors,suggesting potentially important biological relevance of thesemutations. In fact, the two mutations have been demonstrated to conferincreased transcriptional activity on the TERT promoter (Horn et al.2013, Huang et al. 2013). These mutations are not found in normal humansubjects and in the public genetic databases and are, therefore,cancer-specific somatic genetic alterations, further supporting theirpotentially important role in human tumorigenesis. This is consistentwith the previously observed increased telomerase activities in somehuman cancers (Smekalova et al. 2012, Mocellin et al. 2013). Thus, TERTpromoter mutations, by promoting the expression of the catalytic subunitof telomerase in response to ETS transcription factors, probablyrepresent a novel mechanism by which telomerase plays an important rolein human tumorigenesis. Melanomas and follicular cell-derived thyroidcancer share considerably similar genetic backgrounds; for example, theyboth harbor the BRAF V600E mutation with a high prevalence (Davies etal. 2002, Xing 2005a). We were, therefore, prompted to explore TERTpromoter mutations in thyroid cancers in the present study.

Follicular cell-derived thyroid cancer is a common endocrine malignancythe incidence of which, similar to that of melanoma, has been risingrapidly globally in recent years (Jemal et al. 2011, Howlader et al.2012). Follicular cell-derived thyroid cancer can be classified intoseveral histological types (DeLellis et al. 2004), among which the mostcommon types are papillary thyroid cancer (PTC) and follicular thyroidcancer (FTC), which account for 85-90% and 10-15% of all the thyroidcancers respectively (DeLellis et al. 2004, Jemal et al. 2011, Howladeret al. 2012). PTC can be further classified into a few subtypes orvariants, the most common of which include conventional PTC (CPTC),follicular variant PTC (FVPTC), and tall-cell PTC (TCPTC). Othersubtypes of PTC, such as the columnar variant, are rare. Unlike the rarebut rapidly aggressive undifferentiated anaplastic thyroid cancer (ATC;Smallridge et al. 2012), PTC and FTC are indolent differentiated thyroidcancers (DTCs). There is also poorly differentiated thyroid cancer(PDTC), which has aggressiveness between that of DTC and ATC.Para-follicular C-cell-derived medullary thyroid cancer (MTC) isuncommon. Benign thyroid tumors are far more common than thyroidcancers. Various genetic alterations have been identified in thyroidcancers, which, by aberrantly driving various signaling pathways, play afundamental role in thyroid tumorigenesis (Xing 2013). In the presentstudy, we examined TERT promoter mutations in various thyroid tumors toexplore novel genetic alterations in thyroid tumorigenesis.

Materials and Methods

Thyroid tumor tissues, cell lines, and DNA. Genomic DNA was isolatedfrom thyroid tumor tissues and cell lines using standard procedures ofproteinase K digestion, phenol-chloroform extraction, and ethanolprecipitation. Use of human thyroid tissues was based on InstitutionalReview Board-approved protocols and written patient consent was obtainedwhere appropriate. The study included 85 benign thyroid tumors, 257 PTC(consisting of 187 CPTC, 56 FVPTC, 13 TCPTC, and 1 columnar PTC), 79conventional FTC, 8 PDTC, 54

ATC, and 16 MTC samples. Thyroid cancer cell lines included TPC1, C643,Hth7, FTC133, OCUT-1, K1, FB1, BCPAP, SW1736, KAT18, Hth74, and WRO.Their thyroid tumor origins are given in Table 1.

TABLE 1 TERT promoter mutation status of individual thyroid cancer celllines Cell lines TPC1 K1 BCPAP FTC133 WRO C643 Hth7 OCUT1 SW1736 KAT18Hth74 FB1 Tumor PTC PTC PTC FTC FTC ATC ATC ATC ATC ATC ATC ATC originTERT C228T C228T CC229, C228T Wild- C228T C250T C250T C228T C228T C250TC228T promoter 228TT type mutation Zygosity Heter Heter Heter Homo HomoHeter Homo Homo Heter Heter Homo Heter Footnotes: PTC, papillary thyroidcancer; FTC, follicular thyroid cancer; ATC, anaplastic thyroid cancer;“Heter”, heterozygous; “Homo”, homozygous.

Identification of TERT promoter mutations. Standard PCR was carried outfor genetic sequencing to identify TERT promoter mutations. Briefly, afragment of the TERT promoter was amplified by PCR on genomic DNA usingprimers 5′-AGTGGATTCGCGGGCACAGA-3′ (SEQ ID NO:2) (sense) and5′-CAGCGCTGCCTGAAACTC-3′ (SEQ ID NO:3) (antisense). This resulted in aPCR product of 235 bp, containing the sites where mutations C228T andC250T occur in melanomas (Horn et al. 2013, Huang et al. 2013). About40-50 ng of genomic DNA were used in the PCR, which was carried out withan initial denaturation step at 95° C. for 3min, followed by ten cyclesof 95° C. denaturation for 30s, 55° C. annealing for 30s, and 68° C.elongation for 1 min. This was then followed by 30 cycles of the samesettings except for elongation for an additional 5s in each cycle. ThePCR was completed with a final elongation step at 68° C. for 7 min.Following quality confirmation of the PCR products by gelelectrophoresis, sequencing PCR was carried out using a Big Dyeterminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems)and an ABI PRISM 3730 automated next generation genetic analyzer(Applied Biosystems) at the Johns Hopkins' sequencing facility. When amutation was identified by Big Dye sequencing using the sense primer,the reaction was repeated using the antisense primer to confirm themutation.

Identification of BRAF V600E mutation. The BRAF V600E mutation wasanalyzed as described previously (Hu et al. 2006). Briefly, exon 15 ofthe BRAF gene containing the site for the T1799A (V600E) mutation wasPCR-amplified using primers TCATAATGCTTGCTCTGATAGGA (SEQ ID NO:4)(sense) and GGCCAAAAATTTAATCAGTGGA (SEQ ID NO:5) (antisense), resultingin a 212 bp product. The PCR settings included one cycle of 95° C. for5min; two cycles of 95° C. for 1 min, 60° C. for 1 min, and 72° C. for 1min; two cycles of 95° C. for 1 min, 58° C. for 1 min, and 72° C. for 1min; and 35 cycles of 95° C. for 1 min, 56° C. for 1 min, and 72° C. for1 min, followed by an extension step at 72° C. for 5min. After qualityconfirmation by agarose gel electrophoresis, the PCR products weresubjected to Big Dye reaction and sequencing analysis as described abovefor TERT mutations. All the mutations were confirmed using both thesense and antisense primers.

Results EXAMPLE 1 Prevalence of TERT Promoter Mutations in ThyroidCancer Cell Lines and Thyroid Tumors

In FIG. 1, representative electropherograms of the two TERT promotermutations in thyroid cancer cell lines and various primary thyroidcancer tumor samples detected by both sense (FIG. 1A) and antisense(FIG. 1B) primers are shown. In Table 1, the

TERT promoter mutation status of the 12 individual thyroid cancer celllines tested is summarized. Except for the WRO cell line that harboredthe wild-type TERT promoter, all the remaining 11 thyroid cancer celllines examined harbored TERT promoter mutations. PTC and FTC cell linesonly harbored the C228T mutation, while the ATC cell line harbored boththe C228T and C250T mutations. Table 2 summarizes TERT promotermutations found in all the thyroid cancer cell lines and primary thyroidtumors. The two mutations were collectively found in 11 of the 12(91.7%) thyroid cancer cell lines. The C228T mutation was found in 0 of85 (0.0%) benign thyroid tumor, 30 of 257 (11.7%) PTC, 9 of 79 (11.4%)FTC, 3 of 8 (37.5%) PDTC, and 23 of 54 (42.6%) ATC samples. Among thethree variants of PTC, the C228T mutation was found in 4 of 13 (30.8%)TCPTC, 23 of 187 (12.3%) CPTC, and 2 of 56 (3.6%) FVPTC samples. Thesingle columnar PTC sample examined was positive for the C228T mutation.The C250T mutation was not found in the PTC sample, but was found in twoFTC, two ATC, and three ATC cell lines. The two TERT promoter mutationswere mutually exclusive in both thyroid cancer cell lines and thyroidcancer tumor samples and collectively found in 11 of 79 (13.9%) FTC, 25of 54 (46.3%) ATC, and 7 of 7 (100%) ATC cell lines. No TERT promotermutation was found in 16 MTC samples. Three cases had both PTC and ATCin the same thyroid gland, and in each case, both the PTC and ATCharbored the C228T mutation. Three melanoma cell lines (M14, A375, andUACC62) examined harbored the C250T mutation (data not shown), as foundin other melanoma cell lines (Horn et al. 2013, Huang et al. 2013). Allthe TERT mutations in the tumor samples were heterozygous, and some celllines harbored a homozygous C228T or C250T mutation (Table 1). We alsofound a C>T mutation at position chromosome 5: 1 295 229, which isadjacent to the C228T mutation, resulting in a CC>TT tandem mutation inthe BCPAP cell line (FIG. 1A). This is similar to the occasional findingof this tandem mutation in melanomas (Horn et al. 2013, Huang et al.2013). A germline A>C (T>G on opposite strand) mutation at −57 bp fromthe ATG translation start site of the TERT gene was found in familialmelanomas (Horn et al. 2013), but we did not find this mutation in anyof the thyroid tumor samples or cell lines in the present study. We alsodid not find this mutation and other TERT promoter mutations in theperipheral blood DNA of 18 patients with familial PTC from a previousstudy (Xing 2005b).

TABLE 2 TERT promoter mutations in thyroid tumors Mutation MutationCollective C228T C250T mutations Samples n/N (%) n/N (%) n/N (%) ThyroidPTC   3/3 (100.0) 0/3 (0.0)  3/3 (100.0) cancer FTC   1/2 (50.0) 0/2(0.0) 1/2 (50.0) cell ATC   4/7 (57.1)  3/7 (42.9)  7/7 (100.0) linesAll  8/12 (66.7) 3/12 (25)  11/12 (91.7)  Thyroid Benign  0/85 (0.0)0/85 (0)  0/85 (0.0)  tumors tumor PTC CPTC 23/187 (12.3) 0/187 (0.0) 23/187 (12.3)   FVPTC  2/56 (3.6) 0/56 (0.0)  2/56 (3.6)  TCPTC  4/13(30.8) 0/13 (0.0)  4/13 (30.8)  Colum-   1/1 (100.0) 0/1 (0.0)  1/1(100.0) nar All 30/257 (11.7) 0/257 (0.0)  30/257 (11.7)   FTC  9/79(11.4) 2/79 (2.5)  11/79(13.9)  DTC 39/336 (11.6) 2/336 (0.6)  41/336(12.2)   PDTC   3/8 (37.5) 0/8 (0.0) 3/8 (37.5) ATC  23/54 (42.6) 2/54(3.7)  25/54 (46.3)  MTC  0/16 (0.0) 0/16 (0.0)  0/16 (0.0)  Footnotes:PTC, papillary thyroid cancer; CPTC, conventional PTC; FVPTC, follicularvariant PTC; TCPTC, tall cell PTC; FTC, follicular thyroid cancer; DTC,differentiated thyroid cancer (combination of PTC and FTC); PDTC, poorlydifferentiated thyroid cancer; ATC, anaplastic thyroid cancer; MTC,medullary thyroid cancer

Example 2 Association of TERT Promoter Mutations with Aggressive Typesof Thyroid Cancers

CPTC, FVPTC, and TCPTC account for the vast majority of PTC variants.TCPTC is classically known to be more aggressive than CPTC and FVPTC. Asshown in Table 3, TERT promoter mutations were significantly moreprevalent in the TCPTC samples than in the CPTC and FVPTC samples, 30.8%(4/13) in the former vs. 10.3% (25/243) in the latter two (P=0.046, pertwo-tailed Fisher's exact test). TERT promoter mutations were highlysignificantly more prevalent in the ATC samples than in the DTC samples,46.3% (25/54) in the former vs. 12.2% (41/336) in the latter (P=3×10⁻⁸).There was a trend towards a higher prevalence of TERT promoter mutationsin the PDTC samples than in the DTC samples, 37.5% (3/8) in the formervs. 12.2% (41/336) in the latter (P=0.069). Statistical significance wasnot reached, probably due to the relatively small number of PDTCsamples.

TABLE 3 Association of TERT promoter mutations with aggressive thyroidcancers Collective TERT Types of thyroid promoter cancer mutations, n/N(%) P value* TCPTC  4/13 (30.8) 0.046 CPTC + FVPTC 25/243 (10.3) ATC 25/54 (46.3) 3 × 10⁻⁸ DTC 41/336 (12.2) PDTC   3/8 (37.5) 0.069 DT C41/336 (12.2) Footnotes: PTC, papillary thyroid cancer; TCPTC, tall-cellPTC; CPTC, conventional PTC; FVPTC, follicular variant PTC; PDTC, poorlydifferentiated thyroid cancer; DTC, differentiated thyroid cancer(combination of PTC and FTC); ATC, anaplastic thyroid cancer. *Pertwo-tailed Fisher's exact test.

Example 3 Association of TERT Promoter Mutation C228T with BRAF V600EMutation in PTC

BRAF V600E mutation, which activates the MAPK pathway, is the mostcommon mutation in thyroid cancers, particularly in PTC (Xing 2005a).We, therefore, analyzed the relationship between this mutation and TERTpromoter mutation C228T in PTC. As shown in Table 4, TERT promotermutation C228T more commonly occurred in the PTC samples harboring theBRAF V600E mutation than in the PTC samples harboring the wild-type BRAFgene, with a prevalence of 18.3% (19/104) in the former vs. 7.2%(11/153) in the latter (P=0.0094, per two-tailed Fisher's exact test).Conversely, BRAF mutation more commonly occurred in the PTC samplesharboring the TERT promoter mutation than in the PTC samples harboringthe wild-type TERT, 63.3% (19/30) in the former vs. 37.4% (85/227) inthe latter (P=0.0094). Thus, the majority of the TERT promotermutation-positive PTC samples harbored the BRAF V600E mutation. Severalcases of ATC had both BRAF V600E and TERT mutations, but therelationship of the two types of mutations could not be statisticallyanalyzed in this cancer due to the small number of BRAFmutation-positive cases (Table 4).

TABLE 4 Association of TERT promoter C228T mutation with the BRAF V600Emutation in papillary thyroid cancer TERT C228T BRAF V600E Tumormutation n/N (%) P mutation n/N (%) P type BRAF− BRAF+ value* TERT−TERT+ value* PTC 11/153 19/104 0.0094 85/227 19/30 0.0094 (7.2) (18.3)(37.4) (63.3) ATC 20/44 5/10 1.0 5/29 5/25 1.0 (45.5) (50.0) (17.2)(20.0) Footnotes: PTC, papillary thyroid cancer; ATC, anaplastic thyroidcancer. * Per two-tailed Fisher's exact test.

Discussion

The recent discovery of TERT promoter mutations in melanomas is thefirst example, to our knowledge, indicating that mutations in genepromoters may also play an important oncogenic role (Horn et al. 2013,Huang et al. 2013). This represents a novel genetic mechanism in humantumorigenesis. A subsequent report of the existence of TERT promotermutations in other human cancers (Killela et al. 2013) and our report onthe high prevalence of these mutations in bladder cancer andglioblastoma (Liu et al. 2013) suggest that TERT promoter mutations mayplay a huge role in human tumorigenesis. We report here for the firsttime, to our knowledge, that common TERT promoter mutations are also inobserved thyroid cancer.

We found no TERT promoter mutations in para-follicular C-cell-derivedMTC samples, consistent with similar findings in a recent study on 24MTC samples (Killela et al. 2013). However, due to the relatively smallnumber of samples examined, the status of TERT promoter mutations in MTCcannot be definitively concluded. In contrast, in the analysis of alarge cohort of follicular cell-derived thyroid cancer samples in thepresent study, we found a common occurrence of TERT promoter mutationsin both PTC and FTC samples, suggesting a role of these mutations in thetumorigenesis of a subgroup of these DTCs. The lack of TERT promotermutations in benign thyroid tumor samples suggests that these mutationsare malignancy-specific and may be relatively late genetic events alongthe line of thyroid tumorigenesis. Consistent with this idea is thestrikingly higher prevalence of TERT promoter mutations in PDTC and ATCthan in DTCs; PDTC and ATC have partially and completely lostdifferentiation respectively and are the most aggressive thyroidcancers. This raises the possibility that TERT promoter mutations mayplay a particular role in the de-differentiation of DTCs and hence theirconversion to poorly or undifferentiated aggressive thyroid cancers.This possibility is consistent with the finding in three cases in whichco-existing PTC and ATC in the same thyroid gland harbored TERT promotermutation C228T. The prevalence of TERT promoter mutations was extremelyhigh in thyroid cancer cell lines (91.7%), which is in contrast to thelow prevalence of 16% (24/150) in general cancer cell lines from theCancer Cell Line Encyclopedia (Huang et al. 2013), but is similar to thehigh prevalence of 74% (125/168) in melanoma cell lines (Horn et al.2013). This result is again consistent with the idea that TERT promotermutations may play a role in the de-differentiation of thyroid cancercells since thyroid cancer cell lines in culture commonly becomede-differentiated (van Staveren et al. 2007). Interestingly, among thethree common variants of PTC, TCPTC harbored TERT promoter mutationswith the highest prevalence. TCPTC is a relatively uncommon but moreaggressive PTC variant than CPTC and FVPTC

(Xing et al. 2005, Ghossein et al. 2007, LiVolsi 2010). It is possiblethat TERT promoter mutations play a role in the aggressiveness of thisunique PTC variant. This is again consistent with the idea that TERTpromoter mutations may play a role in the development of progression andaggressiveness of thyroid cancers.

As in many other human cancers in which telomerase activities areincreased (Smekalova et al. 2012, Mocellin et al. 2013), increasedtelomerase activities have also been found in thyroid cancers but not innormal thyroid tissues or benign thyroid tumors, suggesting a role ofthis enzyme in thyroid cancer tumorigenesis (Capezzone et al. 2009).Both TERT promoter C228T and C250T mutations create binding sites forETS transcription factors, which subsequently promote the expression ofTERT (Horn et al. 2013, Huang et al. 2013). Thus, TERT promotermutations may contribute to thyroid tumorigenesis by aberrantlypromoting the expression of TERT. Interestingly, some ETS factors aretargets of the MAPK signaling pathway (Janknecht et al. 1995, Whitmarshet al. 1995, Strahl et al. 1996). The MAPK pathway aberrantly activatedby BRAF V600E plays a fundamental role in the tumorigenesis andprogression of PTC (Xing 2013). It is thus possible that TERT promotermutations may join the mechanisms involving the MAPK signaling inthyroid tumorigenesis. Consistent with this hypothesis is theparticularly high prevalence of TERT promoter mutations in BRAF V600Emutation-positive PTC and vice versa found in the present study. Thepreferential occurrence of TERT promoter mutations in BRAF V600Emutation-positive PTC is also consistent with the hypothesis discussedabove that these TERT promoter mutations may play a role in theaggressiveness of thyroid cancers since BRAF V600E mutation-positive PTCis more aggressive than PTC with wild-type BRAF (Xing et al. 2005,2013a). The association between TERT promoter and BRAF V600E mutationscreates a unique mechanism for the amplification of TERT expression, inwhich TERT promoter mutations create binding sites for ETS transcriptionfactors, which, upon activation by BRAF V600E-promoted MAPK signaling,initiate or augment the expression of TERT. Thus, the co-existence ofTERT promoter and BRAF V600E mutations conceivably confers thyroidcancers with a unique survival advantage. New treatments targetingmolecular targets, such as BRAF V600E, are being actively sought andtested for thyroid cancers (Xing et al. 2013b).

The finding of TERT promoter mutations in thyroid cancers opens anexciting possibility for the development of novel therapeutic agentstargeting TERT in thyroid cancer patients. Given the association of TERTpromoter mutations with BRAF V600E mutation and their presumedinteraction through enhancement of the function of ETS transcriptionfactors in regulating the expression of TERT, this therapeutic strategymay be particularly effective in patients with both TERT promotermutations and BRAF V600E mutation.

In summary, herein, we report for the first time, to our knowledge,common TERT promoter mutations in thyroid cancers, which areparticularly prevalent in aggressive types of thyroid cancers and inBRAF V600E mutation-positive PTC. Their occurrence patterns in varioustypes of thyroid cancers suggest that these TERT promoter mutations mayplay a role in the de-differentiation, progression, and aggressivenessof thyroid cancers. The discovery of this novel genetic background ofthyroid cancers opens exciting new opportunities for biological andclinical research of thyroid cancers.

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4. Horn S, Figl A, Rachakonda P S, Fischer C, Sucker A, Gast A, Kadel S,Moll I, Nagore E, Hemminki K et al. 2013 TERT promoter mutations infamilial and sporadic melanoma. Science 339 959-961.

5. Howlader N, Noone A M, Krapcho M, Neyman N, Aminou R, Altekruse S F,Kosary C L, Ruhl J, Tatalovich Z, Cho H et al. (eds) 2012 SEER CancerStatistics Review, 1975-2009 (Vintage 2009 Populations). Bethesda, Md.:National Cancer Institute (http://seer.cancer.gov/csr/1975_2009_pops09/, based on November 2011 SEER data submission, postedto the SEER web site, April 2012).

6. Hu S, Liu D, Tufano R P, Carson K A, Rosenbaum E, Cohen Y, Holt E H,Kiseljak-Vassiliades K, Rhoden K J, Tolaney S et al. 2006 Association ofaberrant methylation of tumor suppressor genes with tumor aggressivenessand BRAF mutation in papillary thyroid cancer. International Journal ofCancer 119 2322-2329.

7. Huang F W, Hodis E, Xu M J, Kryukov G V, Chin L & Garraway L A 2013Highly recurrent TERT promoter mutations in human melanoma. Science 339957-959.

8. Janknecht R, Ernst W H & Nordheim A 1995 SAP1a is a nuclear target ofsignaling cascades involving ERKs. Oncogene 10 1209-1216.

9. Jemal A, Bray F, Center M M, Ferlay J, Ward E & Forman D 2011 Globalcancer statistics. CA: A Cancer Journal for Clinicians 61 69-90.

10. Killela P J, Reitman Z J, Jiao Y, Bettegowda C, Agrawal N, Diaz L AJr, Friedman A H, Friedman H, Gallia G L, Giovanella B C et al. 2013TERT promoter mutations occur frequently in gliomas and a subset oftumors derived from cells with low rates of self-renewal. PNAS 1106021-6026.

11. Liu X, Wu G, Shan Y, Hartmann C, von Deimling A & Xing M 2013 Highlyprevalent TERT promoter mutations in bladder cancer and glioblastoma.Cell Cycle 12 1637-1638.

12. LiVolsi V A 2010 Papillary carcinoma tall cell variant (TCV): areview. Endocrine Pathology 21 12-15.

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Highly Prevelant TERT Promoter Mutations in Bladder Cancer andGlioblastoma

Telomerase reverse transcriptase (TERT) activities are frequentlyupregulated in human cancers, which is thought to be an importantmechanism contributing to human tumorigenesis. Here, we investigatedmutations in the TERT promoter-1,295,228 C>T and 1,295,250 C>T (termedC228T and C250T, respectively) in bladder cancer and glioblastoma. Useof primary bladder cancer and glioblastoma tissues was based oninstitutional review board-approved protocols. Genomic DNA from tumortissues was isolated using standard procedures of protease K digestion,phenol-chloroform extraction and ethanol precipitation. A fragment ofthe TERT promoter was amplified by polymerase chain reaction (PCR) usingprimers 5′-AGTGGATTCGCGGGCACAGA-3′ (SEQ ID NO:2) (sense) and5′-CAGCGCTGCCTGAAACTC-3′ (SEQ ID NO:3) (antisense), resulting in a PCRproduct of 235 bp, which contained the sites of C228T and C250Tmutations. Amplification PCR was performed with an initial denaturationat 95° C. for 3 min, followed by 10 cycles of 95° C. denaturation for 30sec, 55° C. annealing for 30 sec and 68° C. elongation for 1 min. Thiswas followed by 30 cycles of the same settings except for the elongationfor additional 5 sec in each cycle. Quality of PCR products wasconfirmed by gel electrophoresis. Sequencing PCR was performed using aBig Dye terminator v3.1 cycle sequencing ready reaction kit (AppliedBiosystems) and an ABI PRISM 3730 automated next generation geneticanalyzer (Applied Biosystems). Mutations were confirmed by repeatingamplification PCR and using both primers in the Big Dye sequencing.

As summarized in Table 5, we found highly prevalent TERT promotermutations in bladder cancer, bladder cancer cell lines and glioblastoma.C228T was far more common than C250T in all cases. Specifically, wefound C228T in 81% (42/52) of bladder cancer samples and C250T in 4%(2/52) of samples. The two mutations were mutually exclusive in bladdercancer and were collectively found in 85% (44/52) of samples. C228T wasfound in 88% (7/8) of bladder cancer cell lines, and no C250T was foundin these cell dines. We found C228T in 65% (48/74) of glioblastomasamples and C250T in 19% (14/74) of samples. The two mutations were alsomutually exclusive in glioblastomas and were collectively found in 84%(62/74) of samples.

Our study on a large number of cases demonstrated a high prevalence ofTERT promoter mutations in bladder cancer, establishing the commonoccurrence of these mutations in bladder cancer. This prevalence of 85%is unusually high for somatic mutations in any human cancer. We alsofound a high prevalence of TERT promoter mutations in glioblastoma,which like the prevalence of TERT promoter mutations in bladder cancer,is also unusually high for somatic mutations in any human cancer. Ourfinding of the high prevalence of TERT promoter mutations in bladdercancer and glioblastoma helps establish the common occurrence of thisgenetic alteration in the two cancers. Given the high prevalence, it issafe to assume that TERT promoter mutations play an important role inthe tumorigenesis and pathogenesis of bladder cancer and glioblastoma.

With the high prevalence, testing of TERT promoter mutations for thediagnosis of bladder cancer and glioblastoma would be expected to have ahigh sensitivity. As these mutations are only found in cancers, testingof TERT promoter mutations are likely to be also highly specific forbladder cancer and glioblastoma. Both the C228T and C250T mutationscreate an 11-base nucleotide stretch 5′-CCCCTTCCGGG-3′ (SEQ ID NO:1),which contains a consensus binding site, GGAA (in reverse complement),for ETS transcription factors. This has strong functional implicationsfor these mutations; it is expected that these TERT promoter mutationswould lead to upregulation of TERT through the action of ETS transcriptfactors in bladder cancer and glioblastoma. Indeed, the two mutationswere demonstrated to confer upon the TERT promoter increasedtranscriptional activity. As such, these TERT mutations may be novelprognostic factors and therapeutic targets for bladder cancer andglioblastoma. Thus, the discovery of these TERT promoter mutations hasstrong clinical implications for the development of novel diagnostic,prognostic and therapeutic strategies for bladder cancer andglioblastoma as well as other cancers that may harbor these mutations(FIG. 2).

TABLE 5 TERT promoter mutations in bladder cancer and glioblastoma TERTpromoter mutations 1,295,228 1,295,250 C > T (C228T) C > T (C250T)Overall Tumor types n/N (%) n/N(%) n/N (%) Bladder cancer 42/52 (80.77)2/52 (3.85) 44/52 (84.62) Bladder cancer  7/8 (87.50)  0/8 (0.00)  7/8(87.50) cell lines Glioblastoma 48/74 (64.86) 14/74 (18.92) 62/74(83.78)

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TERT Promoter Mutations in Other Cancers

As shown in Table 6, in addition to bladder cancer and brain tumors(glioblastoma), we found frequent TERT promoter mutations also inseveral other human cancers. Use of these mutations can also helpdevelop novel diagnostic, prognostic and therapeutic strategies forthese human cancers.

TABLE 6 TERT promoter mutations in human cancers NEW rs2853669 TERT 228250 242 273 Bladder 36/52 45/52 42/52 2/52 1/52 69.23% 86.53% 80.77% 3.85% 1.92% Brain tumors 22/47 36/47 31/47 5/47 (Glioblastoma 46.81% 76.6% 65.96% 10.64% multiforme) Breast 42/76 4/76 1/76 3/76 55.26% 5.26%  1.32% 3.95% Colon 7/25 0/25   28%    0% Ovarian 3/44 Cancer   7%

We claim:
 1. A method for treating a subject having thyroid cancer comprising the steps of: a. obtaining a biological sample from the subject; b. performing an assay on the sample obtained from the subject to identify a mutation at 1 295 228 C>T (C228T) and 1 295 250 C>T (C250T), corresponding to −124 C>T and −146 C>T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene; c. identifying the subject as having or likely to develop aggressive thyroid cancer if the C228T and/or C250T mutation is identified; and d. treating the subject with one or more treatment modalities appropriate for a subject having or likely to develop aggressive thyroid cancer.
 2. The method of claim 1, wherein the assay of step (b) comprises sequencing of the TERT promoter region comprising −124 and −146 from the translation start site in the promoter of TERT.
 3. The method of claim 1, wherein the assay of step (b) comprises the steps of: i. extracting DNA from the biological sample; ii. contacting the DNA with a primer that specifically hybridizes to the TERT gene; iii. amplifying by polymerase chain reaction (PCR) a region of the TERT gene that comprises −124 and −146 from the translation start site in the promoter of TERT; and iv. sequencing the amplification product to identify the presence of the C228T and/or C250T mutation.
 4. The method of claim 1, wherein the treatment modality for aggressive thyroid cancer comprises thyroidectomy, hemithyroidectomy, radioactive iodine therapy, and combinations thereof
 5. The method of claim 1, wherein the treatment modality comprises administering to the subject a TERT inhibitor.
 6. A method for identifying a subject as having or likely to develop aggressive thyroid cancer comprising the steps of: a. obtaining a biological sample from the subject; b. performing an assay on the sample obtained from the subject to identify a mutation at 1 295 228 C>T (C228T) and 1 295 250 C>T (C250T), corresponding to −124 C>T and −146 C>T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene; c. identifying the subject as having or likely to develop aggressive thyroid cancer if the C228T and/or C250T mutation is identified.
 7. The method of claim 6, wherein the assay of step (b) comprises sequencing of the TERT promoter region comprising −124 and −146 from the translation start site in the promoter of TERT.
 8. The method of claim 6, wherein the assay of step (b) comprises the steps of: i. extracting DNA from the biological sample; ii. contacting the DNA with a primer that specifically hybridizes to the TERT gene; iii. amplifying by polymerase chain reaction (PCR) a region of the TERT gene that comprises −124 and −146 from the translation start site in the promoter of TERT; and iv. sequencing the amplification product to identify the presence of the C228T and/or C250T mutation.
 9. The method of claim 6, further comprising the step of administering a treatment modality appropriate for a subject having or likely to develop aggressive thyroid cancer.
 10. The method of claim 9, wherein the treatment modality for aggressive thyroid cancer comprises thyroidectomy, hemithyroidectomy, radioactive iodine therapy, and combinations thereof.
 11. The method of claim 9, wherein the treatment modality comprises administering to the subject a TERT inhibitor. 